Insights into the Role of a Disordered N-Terminus in the Functional Regulation of CLK1 Kinase

SR proteins are a family of splicing factors that plays important roles in regulating alternative splicing. The function of SR proteins is heavily regulated by the extent of phosphorylation of its C-terminal RS domain. Cdc-2 like kinases (CLKs) are known to hyper-phosphorylate the RS domains and control the splicing functions of SR proteins. CLKs have a folded kinase domain and an N-terminus that is predicted to be disordered. The N-terminus plays vital roles in regulating the function of CLK1 in various aspects. The studies presented here elucidate how the N-terminus modulates three different aspects of CLK1 function: sub-cellular localization, self-association to form oligomers, and substrate phosphorylation. Although prior studies had shown that the N-terminus was important for modulating the nuclear import of CLK1, the mechanism for this change in subcellular localization had largely been uninvestigated. Here, we show that CLK1 lacks a short, classical Nuclear Localization Sequence (NLS), indicating that the nuclear import is not mediated by the classical importin / system. Instead, we show that CLK1 enters the nucleus by forming a complex with its physiological substrate SRSF1, an SR protein prototype, in the cytoplasm and transportin SR-2 (TRN-SR2) imports the kinase-substrate complex into the nucleus. Previous studies from our laboratory had shown that the N-terminus induces oligomerization of CLK1, which helps the kinase select its physiological substrates over non-physiological ones. The nature of the interactions underlying this oligomerization was investigated and our results show that CLK1 oligomerization is driven not only by self-association of the N-terminus (N-N interactions) but also by interactions between the N-terminus and the kinase domain (N-K interactions). While interactions between the N-termini are mediated solely by aromatic residues, interactions between the N-terminus and kinase domain are electrostatic in nature. Lastly, we also inves